Regulatory T cells (Tregs) are critical to the maintenance of self-tolerance, prevention of autoimmunity, and inhibition of inflammatory diseases; however, they can also function to limit sterilizing immunity against pathogens and favorable anti-cancer responses. Therefore, clarifying the mechanisms of Treg-mediated suppression in the context of cancer will aid in the rational design of new therapies. Our lab has recently identified a novel suppressive role for the IL35 heterodimer in Tregs. IL35 is a member of the IL12 family comprised of the IL12a and Ebi3 subunits and is preferentially expressed by Tregs. IL35 is known to induce infectious tolerance, a process by which conventional T effector cells become a highly suppressive, IL35- producing population (iTr35s). The tumor microenvironment contains suppressive factors such as TGF-, IDO, IL10, myeloid-derived suppressor cells, and Tregs, which inhibit antigen-specific cytotoxic lymphocyte responses. Depletion or inhibition of Tregs and suppressive signaling pathways has been shown to decrease tumor burden and enhance patient outcome in both animal models and the clinic. We hypothesize that the regulatory triad of IL35, Tregs, and iTr35s contributes critically to the tumo microenvironment, and that elimination of IL35 will enhance tumor specific immunity. To test our hypothesis, we have proposed two separate Aims. For the first Aim, to determine if IL35-mediated suppression contributes to tumor growth, we will primarily utilize the weakly-immunogenic B16 melanoma model in syngeneic, reporter, and/or Rag1-/- hosts. We will monitor B16 growth, animal survival, and study molecular signatures of tumor bearing mice in the context of IL35 neutralization. IL35 neutralization studies will be extended to include a more rigorous and clinically-relevant genetically-induced model of non-small cell lung cancer. In the second Aim, we will determine the expression pattern, kinetics, and suppression requirements of IL35 in tissues of tumor-bearing mice. We will use our newly-developed cytokine capture assay and several reporter mice, including Ebi3Tom and Foxp3GFP, to elucidate the expression pattern and kinetics of IL35 at the single-cell level. Finally, Treg-specific IL35 knockout mice wil be used to determine if IL35 secretion from Tregs is required to mediate suppression of anti-tumor immunity. Research on these Aims should yield exciting knowledge that will advance the field and hopefully prove to be therapeutically beneficial.